Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants.

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO- ), hypochlorous acid (HOCl), hydrogen peroxide (H2 O2 ), and peroxymonocarbonate (HOOCO2 - ) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid (MpC-BA) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO- or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO- to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one (MpC-OH). However, peroxynitrite-specific product (MpC-H) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl (MpC-OHCl). H2 O2 slowly oxidizes MpC-BA. However, the addition of NaHCO3 increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.

Water-soluble cationic boronate probe based on coumarin imidazolium scaffold: Synthesis, characterization, and application to cellular peroxynitrite detection.

Peroxynitrite (ONOO-) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. Here, the development of a new water-soluble and cationic boronate probe based on a coumarin-imidazolium scaffold (CI-Bz-BA) for the fluorescent detection of ONOO- in cells is reported. The chemical reactivity of the CI-Bz-BA probe toward selected oxidants known to react with the boronate moiety was characterized, and the suitability of the probe for the direct detection of ONOO- in cell-free and cellular system is reported. Oxidation of the probe results in the formation of the primary hydroxybenzyl product (CI-Bz-OH), followed by the spontaneous elimination of the quinone methide moiety to produce the secondary phenol (CI-OH), which is accompanied by a red shift in the fluorescence emission band from 405 nm to 481 nm. CI-Bz-BA reacts with ONOO- stoichiometrically with a rate constant of ∼1 × 106 M-1s-1 to form, in addition to the major phenolic product CI-OH, the minor nitrated product CI-Bz-NO2, which is not formed by other oxidants tested or via myeloperoxidase-catalyzed oxidation/nitration. Both CI-OH and CI-Bz-NO2 products were also formed in the presence of cogenerated fluxes of nitric oxide and superoxide radical anion produced during decomposition of a SIN-1 donor. Using RAW 264.7 cells, we demonstrate the ability of the probe to report endogenously produced ONOO-via fluorescence measurements, including plate reader real time monitoring and two-photon fluorescence imaging. Liquid chromatography/mass spectrometry analyses of cell extracts and media confirmed the formation of both CI-OH and CI-Bz-NO2 in macrophages activated to produce ONOO-. We propose the use of a combination of real-time monitoring of probe oxidation using fluorimetry and fluorescence microscopy with liquid chromatography/mass spectrometry-based product identification for rigorous detection and quantitative analyses of ONOO- in biological systems.

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite.

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO-), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H2O2 to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO-. The ability of the BC-BA probe to detect ONOO- was measured using both authentic ONOO- and the system co-generating steady-state fluxes of O2•- and •NO. BC-BA is oxidized by ONOO- to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.

Two-photon fluorescent probe for cellular peroxynitrite: Fluorescence detection, imaging, and identification of peroxynitrite-specific products.

A new naphthalene-based boronate probe, NAB-BE, for the fluorescence-based detection of inflammatory oxidants, including peroxynitrite, hypochlorous acid, and hydrogen peroxide, is reported. The chemical reactivity and fluorescence properties of the probe and the products are described. The major, phenolic oxidation product, NAB-OH, is formed in case of all three oxidants tested. This product shows green fluorescence, with a maximum at 512 nm, and can be excited either at 340 nm or in the near infrared region (745 nm) for two-photon fluorescence imaging. Peroxynitrite is the fastest of the oxidants tested and, in addition to the phenolic product, leads to the formation of a nitrated product, NAB-NO2, which can serve as a fingerprint for peroxynitrite. The probe was applied to detect peroxynitrite in activated macrophages using fluorimetry and two-photon fluorescence microscopy, and both NAB-OH and NAB-NO2 products were detected in cell extracts by liquid chromatography-mass spectrometry. The combined use of fluorometric high-throughput analyses, fluorescence imaging, and liquid chromatography-mass spectrometry-based product identification and quantitation is proposed for most comprehensive and rigorous characterization of oxidants in biological systems.